Virtually unique among somatic tissues, the brain and spinal cord lack a lymphatic system. Despite the high metabolic activity and fragility of neural tissue, there exists no effective understanding of the means by which interstitial fluid and waste products are removed from the CNS. Our preliminary analysis, based on in vivo two-photon imaging, shows that low molecular weight tracers delivered to the CSF circulate surprisingly rapidly through the mouse brain, and do so along a surprising anatomical route. This consists of a para-arterial inflow path, a trans-glial intra-parenchymal path of interstitial flow, and a para-venous outflow path. Within the intra-parenchymal pathway, astrocytes support convective fluid currents through the brain interstitial space, as deletion of the astrocytic water channel AQP4 sharply reduces overall tracer flow along these routes. Given the continuous movement of fluid supported by this pathway, and its critical dependence upon astrocytic water transport, we propose that this system - which we designate here the 'glymphatic system' - subserves a function homologous to the peripheral lymphatic system, and is essential for the clearance of metabolic waste products from the CNS. Aim 1 will use 2-photon in vivo microscopy to assess the spatial dynamics and temporal kinetics of fluorophore-tagged tracer clearance. By systematically comparing the effect of modifications of molecular sizes or surface charge upon tracer clearance, we will define the basic transport properties of the glymphatic system. Aim 2 will extend the preliminary observation that aged mice exhibit a striking decline in glymphatic system function, and evaluate the role of age-related suppression of arterial wall pulsation and resulting reduced convective inflow along the para-arterial path and global glymphatic function. Aim 3 proposes that induced knock-out of either astrocytic AQP4 water channels or gap junctions (Cx43/Cx30) will slow parenchymal convective fluid flow and globally suppress tracer clearance. Aim 4 tests the proposition that suppression of trans-astroglial fluid movement resulting from AQP4 or Cx43/Cx30 deletion will slow clearance of exogenous A and thereby potentiate age-related amyloid plaque formation. We predict that slowing astrocytic parenchymal fluid flow will accelerate paravascular amyloid deposition, which in a feed-forward manner will further reduce the efficiency of clearance of waste products by the glymphatic system. To our knowledge, these studies represent the first attempt to systematically define the mechanisms involved in the clearance of metabolic waste products from the brain on a whole-organ level. Two-photon imaging of through chronic cranial windows will allow imaging of tracer clearance in real time, whereas transgenic mice with inducible deletion of key astroglial membrane proteins will establish the functional role of astrocytes in glymphatic transport. Combined, these studies will provide fundamental insight into the mechanisms contributing to age-related accumulation of neurotoxic metabolic waste products and define novel, and likely highly important, functional properties of astrocytes. PUBLIC HEALTH RELEVANCE: The proposed studies will define the function of astroglia fluid transport in clearance of metabolic waste.